G. Zambonin

Biomaterials in orthopaedic surgery: effects of different hydroxyapatites and demineralized bone matrix on proliferation rate and bone matrix synthesis by human osteoblasts

The effects of different biomaterials, used in orthopaedic surgery for bone substitution and/or prosthesis coating to improve fixation and durability of prosthetic implants, have been studied in vitro on cell growth and bone matrix synthesis by human osteoblasts. The materials were a bovine collagen matrix (Osteovit, B. Braun Melsungen AG, Melsungen, Germany) and two hydroxyapatite (Ceros 80, Robert Mathys Co, Instrumentenfabrik Bettlach, Germany and Ostilit, Howmedica International, Staines House, UK). Cell proliferation and bone matrix synthesis were assessed by incorporation of [3H]thymidine and [3H]proline, respectively. Cell viability in the presence of the materials was also morphologically controlled using phase-contrast microscopy. Exposure to Osteovit caused increased proliferation of human osteoblasts, whereas both Ostilit and Ceros 80 induced a decreased cell growth. Osteoblast bone matrix synthesis was increased by all the materials tested.

Alendronate Reduces Adhesion of Human Osteoclast-like Cells to Bone and Bone Protein-Coated Surfaces

Bisphosphonates (BPs) are potent inhibitors of bone resorption and are therapeutically effective in disease of increased bone turnover, but their mechanism(s) of action remain to be elucidated. Using as experimental model human osteoclast-like cell lines derived from giant cell tumors of bone, extensively characterized for their osteoclast features, we investigated the adhesive properties of osteoclasts on bone slices and on different proteins of the extracellular matrix in the presence of BPs. Adhesion assays using bone slices pretreated with ALN, at the established active concentration, showed that, although the morphology of osteoclasts plated onto pretreated bone slices was not modified, the number of adherent cells was reduced by the treatment of about 50% vs. controls. The effect of ALN on the adhesion of osteoclast-like cells onto specific extracellular matrix proteins, such as bone sialoprotein-derived peptide, containing the RGD sequence, conjugated to BSA (BSP-BSA) and fibronectin (FN), was also tested. In the case of FN the treatment with ALN of protein-coated wells did not modify the percentage of cell adhesion compared with the control, whereas onto BSP-BSA the presence of ALN significantly reduced adhesion of about 40–45%, suggesting that the inhibitory effect of ALN on cell adhesion could probably be due to the interference with receptors specifically recognizing bone matrix proteins as αVβ3 integrins. Furthermore, ALN induced Ca-mediated intracellular signals in osteoclasts, triggering a 2-fold increase in intracellular calcium concentration.

Response of human osteoblasts to polymethylmetacrylate In vitro.

Abstract. The effects of a polymethylmetacrylate (PMMA) powder with a diameter between 0.5 and 25 μm have been studied in vitro on several human osteoblast populations obtained from different sources. Parameters of cell activity such as cell growth, collagen synthesis, osteocalcin, and interleukin-6 (IL-6) production have been evaluated. Cell proliferation and collagen synthesis were inhibited after exposure to bone cement, whereas osteocalcin and IL-6 production were stimulated. These results suggest that PMMA particles could affect osteoblast activity in a way that could contribute, together with other factors, to periprosthetic osteolysis through two different pathways: a reduced periprosthetic bone formation due to the reduced osteoblast proliferation and collagen synthesis, and an osteoblast-mediated activation of osteoclastic bone resorption as suggested by the increased osteocalcin and IL-6 synthesis. In fact, osteocalcin has been demonstrated to have a role in osteoclast recruitment to bone surfaces, and IL-6 is known to induce osteoclastogenesis and to directly stimulate bone resorption.

Hydroxyapatite coated with hepatocyte growth factor (HGF) stimulates human osteoblasts in vitro

We have studied in vitro the effect of a hydroxyapatite (HA) tricalcium phosphate material coated with hepatocyte growth factor (HA-HGF) on cell growth, collagen synthesis and secretion of metalloproteinases (MMPs) by human osteoblasts. Cell proliferation was stimulated when osteoblasts were incubated with untreated HA and was further increased after exposure to HA-HGF. The uptake of [3H]-proline was increased after treatment with HA. When osteoblasts were exposed to HA-HGF, collagen synthesis was increased with respect to HA. The secretion of MMPs in control cells was undetectable, but in HA and HA-HGF cells MMP 2 and MMP 9 were clearly synthesised. Our results suggest that HA can promote osteoblast activity and that HGF can further increase its bioactivity.

Detection of collagen synthesis by human osteoblasts on a tricalcium phosphate hydroxyapatite: An X‐ray photoelectron spectroscopy investigation

The effect of a hydroxyapatite-tricalcium phosphate (HA) material on collagen synthesis by human osteoblasts was investigated using X-ray photoelectron spectroscopy (XPS). To this aim, thin HA slices were exposed to osteoblasts harvested from three different patients, for 20 days and then analyzed by XPS. Platinum plates were also exposed to the cells for comparison, and control tests were performed on both materials using cell-free media. XPS analysis supported by standard spectra of some polyaminoacids and of collagen deposited on HA suggested that a deposition of collagen occurred on the HA slices in the presence of osteoblasts. On the other hand, only an aspecific deposition of proteins was observed on platinum and when cell-free media were used. These data were confirmed evaluating collagen synthesis by [(3)H]proline incorporation of osteoblasts exposed to HA.

Human Osteoclast-Like Cells from Giant Cell Tumors of Bone: A New Tool for Investigating Bone Resorption and Osteoclast Biology

The effort of culturing human osteoclasts for studies on the resorptive activity have been undertaken without success in many laboratories because of the difficulty of having a sufficient number of differentiated cells from both surgery fragments or generated in vitro. To overcome this difficulty we have extensively studied the cell populations harvested from several human giant cell tumors of bone. In the literature, discordant reports concerning these tumors can be found ,some indicating a stromal nature for the transformed cells and the osteoclasts simply as reactive cells in the tissue [1, 2]. Other authors have shown that the populations obtained maintained multinuclear elements after passages [3] or that the cells, although mononucleated, were responsive to calcitonin [4]. This discrepancy can be explained if the tumors classified as osteoclastoma are two different kinds: one in which the transformed element is a stromal cell, endowed with the capability of recruiting osteoclasts and another in which the transformed cell belongs to the osteoclastic lineage. With this aim, cells from giant cell tumors of bone (GCT) have been characterized from surgical samples obtained in the course of the surgical removal of the tumor, with the informed consent of the patient. The morphological features of these cultures, together with their total and tartrate-resistant acid phosphatase activity, bone resorbing capability, and response to calciotropic hormones have been evaluated. Among all the tumors examined (16 up to now), three with a complete panel of osteoclastic features were found, whereas other three, although mononucleated , were responsive to calcitonin and capable of bone resorption. The cultures from GeT 23, 24, and 29 contained monoand multinuclear cells maintained after several passages (up to 13th until now). Multinuclear cells were originated by both fusion or endomitosis. Tartrate-resistant alkaline phosphatase (TRAP) activity histochemically evaluated was present at different degrees, but could be biochemically evaluated and always ranged around 20% of the total AP activity. Treatment with calcitonin approximately doubled the intracellular content of cAMP. Bone resorption was assayed both biochemically, utilizing prelabeled bone fragments, and morphologically, measuring the formation of resorption pits on bone slices. With both techniques, resorption was evident and in the presence of calcitonin was significantly reduced [5]. Interactions with connective tissue or bone matrixderived proteins were also studied. GCTs adhered and spread on fibronectin, osteopontin, BSPII, as in the pres-

Cellular Basis of Bone Resorption

Bone remodeling is a finely tuned process that lasts throughout one’s life. Old bone is removed, and new bone is formed according to the mechanical and metabolic needs of the body. Osteoblasts and osteoclasts are the cell types that take part in this complex task. The osteoblasts are very possibly, together with the osteocytes, responsible for selecting the area that is to be removed, while the osteoclasts are the cells that actually carry out the resorption. Newly formed osteoblasts are immediately thereafter recruited to repair and/or reorganize the tissue. This chapter describes principally the origin of the osteoclasts, the molecular mechanisms of bone resorption, and the current hypothesis about the coupling mechanism that joins osteoclast and osteoblast activity.

Hepatocyte growth factor links osteoclast-osteoblast actyvity through an autocrine-paracrine signaling

OSTEOBLAST ACTYVITY THROUGH AN AUTOCRINE PARA CRLNE SIGNALING M. Grano, F. Galimi, G. Zambonin, S. Colucei, G. Mori, E. Cottone, P.M. Comoglio, A. Zambonin 7_,allone. Institute of Human, I Orthopaedic Clinic, University of Bari Medical School, and Dept. of Biomedical Science & ontology, University of Torino. Hepatocyte Growth Factor CRGF) is a disulfide-linked haterodimer secreted by cells of mesodermal origin as an inactive single chain precorsor, that is activated to mature form by extraenllular umkinase and related protcases. The HGF receptor, the tyrosine kinase encoded by the c-MET protoeneogane (plg~r) , has been recently detected in a variety of epithelial er raesedermal derived cells. We examined if pl9lY ~ r was expressed in primary human osteoblasts and osteoclasta as well as in tumor derived osteoclast-like cell lines (GCTs) by immunofluorescence and western blotting analysis, finding a high level of expression in both cell ty~s. The treatment with HGF stimulated receptor kinase activity suggasling a potemial role of HGF in bone remodeling, thus the possible biological effects of HGF on osteoblast and osteoclast activity were evaluated. A motility response was highly evident in osteoclasts at concentration of 5 ng/ml compared with negative control, while no effect was found in osteoblasts. A dose-response stimulation of cell proliferation, evaluated by 3H-thymidinr incorporation, was found both in osteeclasts where a concentration of 7.5 ng/ml HGF induced a four-fold increase in 3Hthymidine incorporation, and in osteoblasts where a three-fold increase was already found at 2 ng/ml. The effects of HGF on ostooblast parameters as alkaline phosphatase activity and osteocalcin production were evaluated, demoostrating an inhibitory effect already visible at low dosages. lutraecllular signals as variation in intracellniar calcium concentration 2+ 2+ [Ca ]i were also iovestigated, showing an HGF induced [Ca li increase in osteeclasts and in GCT cells and ne effects io esteoblasls, c-Src kinase activity was evaluated following stimulation by the HGF iu osteoclast-like GCT cells. Results showed a 2-fold increase in pp60-c-Sre kinase in the cells treated with HGF. Moreover results obtained with western blot technique indicate that osteoclasts can produce HGF suggesting an autucrine-garacrine activity and the possible identification of HGF as a coopling factor of osteoclast and osteoblast activily. These data, together with the already demonstrated angiogenic activity of HGF, lel us hipothesizr a prominent role of this growth factor during skelelogenesis and bone remodeling